Covalent immobilization of delipidated human serum albumin on poly(pyrrole-2-carboxylic) acid film for the impedimetric detection of perfluorooctanoic acid
Graphical abstract
Introduction
Electrochemical sensors and biosensors are answering the increasing need of portable tools for the semi-quantitative detection of environmental contaminants (EC) by direct electrochemical fingerprinting or indirect sensing strategies [1]. In the broader context of EC, per- and polyfluoroalkyl substances (PFAS) represent a class of chemicals in continuous expansion (see emerging PFAS in [2], [3]). PFAS and particularly perfluorooctanoic acid (PFOA) are still subjected to extensive toxicological studies to clarify their effects on the ecosystem and human health [4]. Nonetheless, previous studies already claimed their tendency to undergo bioaccumulation in vegetal and animal tissues [5] suggesting the need of large-scale, strict monitoring plans [6] and efficient water treatments [7]. For these reasons, there is an urgent demand of novel sensors for natural and industrial water monitoring, which needs to be rapid, user-friendly, robust and sensitive enough to reach the legislation limits of such EC. Electrochemical biosensors have been regarded as one of the most promising alternative to meet analogous demands in several other fields, such as point-of-care testing [8] and food safety [9].
Concerning the detection of PFOA, only few examples of electrochemical sensors have been reported up to now, mainly potentiometric [10], electrochemiluminescence [11] and photoelectrochemical [12] ones. These sensors are all based on biomimetic receptors such as the molecularly imprinted polymers (MIP) employed also in PFOA removal [13] and non-electrochemical sensing devices [14]. However, also bioreceptors can play an important role in PFOA sensing, as showed by the immunosensor developed by Cennamo et al. [15]. So far, protein-based bioreceptor have not been considered, although serum proteins-PFAS interactions were largely investigated [16]. In particular, PFOA capability to interact with albumin (thanks to PFOA fatty-acid mimic behaviour) was clearly stated in numerous toxicological studies [17], [18] giving the opportunity to use these common proteins as a bioreceptors for PFOA sensing. Moreover, albumin-based electrochemical sensors have already showed good performances in the highly selective and sensitive detection of small molecules [19] as well as larger targets [20]. Albumin is non-electroactive and often combined with impedimetric affinity-based sensors, where the bioreceptor is first immobilized on the electrode surface, detecting the protein-target interaction as a localized change in the electrode-solution interface [21]. However, the immobilization of the bioreceptor is usually a bottleneck in the development of new sensors, particularly at screen printed electrode. To overcome this issue, other surface modifiers are often included to guarantee the stability of the immobilization as well as the electrode surface conductivity and consequently the device sensitivity. Zamani et al. [22] recently reviewed the preeminent role of conductive electropolymerized polymers, especially pyrrole and its derivatives, that were extensively studied and applied as electrode modifiers [23]. Among the pyrrole derivates, pyrrole-2-carboxylic acid (Py-2-COOH) was less employed in electroanalytical applications [24], [25], [26], even possessing many sought-after characteristics compared to the parent compound pyrrole, such as the presence of the carboxylic acid functional groups which can be used to couple amide-bearing molecules to the surface via EDC/NHS chemistry. The complete characterization of the electrochemical polymerization pathway of Py-2-COOH has been previously reported [27]. The results obtained by Foschini et al. [27] allowed to conclude that the electropolymerization mechanism is very similar to the one of pyrrole, as reported by Dias et al. [28]. The final polymeric chain will present a torsion angle between subsequent monomeric units of 74°, compared to 54° of pyrrole [27]. Thus, the electropolymerization conditions already reported and studied for pyrrole can also be applied to the polymerization of Py-2-COOH. Considering the specific electron transfer properties of polypyrrole, the conductivity of the resulting film is influenced by the electrolyte in which the electropolymerization takes place [28]. The polypyrrole film is doped with about 20–30% of anions coming from the electrolyte, and the conductivity is linked to the exchange of trapped anions between the film and the solution [29]. Moreover, acid anions increase the conductivity, while basic ones decrease it [30]. Also the size of the anion is important for the growth of the film and its performances, such as stability to overoxidation [31]. All these parameters need to be taken into account to design suitable poly(Py-2-COOH) modification protocol compatible with graphite screen-printed electrodes (G-SPE).
Aiming to obtain stable and conductive poly(Py-2-COOH) on the electrode surface for the covalent immobilization of biomolecules, we first optimized the main electropolymerization parameters based on previously reported data [32], [33], [34]. In particular, the comparison of two different electrolytes, one organic (SDS) and another inorganic (NaClO4), allowed understanding the influence of the anions on the conductivity of the obtained films at G-SPE. The optimized poly(Py-2-COOH) was then tested in an impedimetric biosensing platform. Delipidated human serum albumin (hSA) was selected as bioreceptor for PFOA sensing. The delipidation treatment, removing the fatty acids embedded in the protein, increase the available bindings site for PFOA [35], improving also the batch-to-batch reproducibility of the bioreceptor. The binding event was followed by electrochemical impedance spectroscopy (EIS) and the changes in the hSA-PFOA complex conformation were verified by SEC-SAXS (Size Exclusion Chromatography-Small-Angle X-ray Scattering). This proof-of-concept application confirmed the possibility to develop simple biosensing tools at poly(Py-2-COOH) modified G-SPE for small molecules detection, such as PFOA. This detection strategy for PFOA, based on protein bioreceptor, offers two main advantages over the already reported electrochemical biosensors: it is label-free, not requiring any label or co-reactant to perform the detection and has the potential to be an easy, fast and robust method of fabrication, based on disposable screen printed electrode and not requiring complex instrumentations or time consuming modification protocols.
Section snippets
Materials and methods
Pyrrole-2-carboxylic acid (99%) (Py-2-COOH), perfluorooctanoic acid (PFOA, ≥96%), perfluorooctanesulfonic acid potassium salt (PFOS, ≥98%) and hydroxylamine were purchased from Sigma–Aldrich Ltd (Belgium). 1-ethyl-3-(3-dimethylamino) propyl carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS) were purchased from TCI (Europe). Highly purified delipidated human serum albumin (hSA) was obtained following the protocol reported in [36]. All the other reagents were of analytical grade
Electropolymerization optimization
Surface modifications of screen printed electrodes may represent a challenging task and often protocols optimized for other type of electrodes (i.e. bulk macroelectrodes) are not directly compatible with SPE [46]. Modification protocols for SPE have to be straightforward and fast, while maintaining the performances of the bulk macro electrodes. In this study, the monomer electropolymerization was carried out by CV in aqueous solution avoiding organic solvents that might affect the stability of
Conclusion
Herein, the electropolymerization of a conductive poly(Py-2-COOH) modifier was optimized at G-SPE in 0.1 M SDS. The choice of this electrolyte assured reproducible and conductive polymer films on the screen-printed surface. Delipidated hSA was then covalently immobilized at the modified electrode via EDC/NHS coupling and the resulting modification was characterized by EIS. The obtained biosensing platform was tested in presence of increasing concentrations of PFOA and the changes at interfacial
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We gratefully acknowledge Gert Nuyts for the SEM measurements and FWO for funding the analytical equipment.
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These authors contributed equally to this work and should be considered co-first authors.